Retroviruses, such as HIV-1 that causes AIDS, have an RNA genome that is reverse-transcribed into a linear viral DNA upon entering the host cell. Integration of this viral DNA into host's chromosome is an essential step in the lifecycle of retroviruses, and is carried out by the virally encoded integrase (IN) protein. Retroviral INs function as a tetramer and catalyze processing of the blunt-ended viral DNA ends as well as subsequent concerted insertions of these processed ends into the backbones of a target DNA. While the chemistry of the IN-catalyzed reactions is well understood, much less is known about how IN carries out these reactions. Key unanswered questions include;Why do the viral DNA 3'-ends need to be resected prior to the joining of viral and target DNA strands? What are the roles of non-catalytic subunits within the tetrameric IN complex? How are the reactions at two viral DNA ends coordinated? How are retroviral INs different from DNA transposases that can perform both integration and excision? Despite the high medical relevance of retroviral IN, no crystal or NMR structure is available for any IN-DNA complex or a full-length three-domain IN protein responsible for the concerted integration reaction. The lack of structural information has been a significant limitation in our mechanistic understanding of the IN-catalyzed reactions. The goal of this proposed research is to obtain the critically needed three-dimensional structural information on the functional multimeric IN assembly and IN-DNA complexes. Using x-ray crystallography we will determine the structure of an engineered three-domain IN protein that we have crystallized. We will also pursue crystal structures of INs from the human pathogen HIV-1 and its model system Rous Sarcoma Virus (RSV) in complex with DNA. IN is an emerging anti-HIV drug target, with the first FDA-approved HIV-IN inhibitor raltegravir in clinical use. The structural information obtained through this research will help understand the bases for drug-resistance mutations and design new inhibitors. In addition, our research may contribute to development of an engineered IN system with sequencing-specific integration properties for safer gene-therapy. During the initial 2-year R21 (ARRA) phase, we will focus on the structure determination of an intact 3-domain IN as proposed in our aim1.